Dronpa_JPC_resub_26_8_13.doc

Dronpa_JPC_resub_26_8_13.doc - 1 Protein Photochromism...

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1 Protein Photochromism Observed by Ultrafast Vibrational Spectroscopy Andras Lukacs, 1 Allison Haigney, 2 Richard Brust, 2 Kiri Addison, 1 Michael Towrie, 3 Gregory M. Greetham, 3 Garth A. Jones, 1 Atsushi Miyawaki, 4 Peter J. Tonge 2 * and Stephen R. Meech 1 * 1. School of Chemistry, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, UK. 2. Department of Chemistry, Stony Brook University, Stony Brook, New York, 11794, USA. 3. Central Laser Facility, Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX, UK 4. Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan *Authors for correspondence ([email protected]; [email protected]) † Present address: Department of Biophysics, Medical School, University of Pecs, Hungary ‡ Present address: The Wistar Institute, Philadelphia, PA 19104 USA
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2 Abstract Photochromic proteins, such as Dronpa, are of particular importance in bioimaging, and form the basis of ultraresolution fluorescence microscopy. The photochromic reaction involves switching between a weakly emissive neutral trans form of the chromophore (A) and its emissive cis anion (B). Controlling the rates of switching has the potential to significantly enhance the spatial and temporal resolution in microscopy. However, the mechanism of the switching reaction has yet to be established. Here we report a high signal-to-noise ultrafast transient infra-red investigation of the photochromic reaction in the mutant Dronpa2, which exhibits facile switching behavior. In these measurements we excite both the A and B forms and observe the evolution in the IR difference spectra over hundreds of picoseconds. Electronic excitation leads to bleaching of the ground electronic state and instantaneous (sub picosecond) changes in the vibrational spectrum of the protein. The chromophore and protein modes evolve with different kinetics. The chromophore ground state recovers in a fast non-single-exponential relaxation, while in a competing reaction the protein undergoes a structural change. This results in formation of a metastable form of the protein in its ground electronic state (A’), which subsequently evolves on the time scale of hundreds of picoseconds. The changes in the vibrational spectrum that occur on the sub-nanosecond time scale do not show unambiguous evidence for either proton transfer or isomerization, suggesting that these low yield processes occur from the metastable state on a longer time scale and are thus not the primary photoreaction. Formation of A’, and further relaxation of this state to the cis anion B, are relatively rare events, thus accounting for the overall low yield of the photochemical reaction. Keywords: Dronpa, Dronpa2, photoswitching, cis-trans, time resolved infrared spectroscopy, ultrafast
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3 Introduction Photochromic proteins, of which Dronpa is the prototypical example, 1-2 form an important subset of the green fluorescent protein (GFP) family. Dronpa is repeatedly photoswitchable between a green
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